The fermentative production of tryptophan by microorganisms from inexpensive carbohydrates is highly desirable. The production of tryptophan by using artificially mutated microorganisms has been known for some time. Classically mutated microbes for tryptophan production include Brevibacterium (described in U.S. Pat. No. 3,700,536), Corynebacterium (ATCC 21851), Bacillus subtilis (described in U.S. Pat. No. 4,363,875), and Enterobacter (described in U.S. Pat. No. 4,439,627).
The use of recombinant DNA technology for the construction of microorganisms for production of tryptophan has been described. These descriptions include Corynebacterium or Brevibacterium (described in U.S. Pat. No. 5,447,857) and Bacillus (described in U.S. Pat. No. 4,588,687). The use of recombinant DNA techniques for the construction of E. coli strains for tryptophan production has also been described in U.S. Pat. No. 4,371,614. In this patent, the maximum production of tryptophan is about 230 ppm or 230 mg/l. However, this amount of tryptophan production is too low for a commercially feasible production strain.
Work to improve tryptophan production was described in (WO 87/01130) and in Applied and Environmental Microbiology (1991) 57: 2995-2999. These documents describe a two plasmid system developed by Stauffer Chemical Co. containing a feedback resistant AS gene (trpE) isolated from Serratia marcessens and a feedback resistant DS gene (aroG) from E. coli. These plasmids also contained the rest of the trp operon trpDCDA from E. coli coding for the APRT, PRAI, InGPS and TS enzymes respectively. One of the plasmids also contained the serA gene used to maintain the plasmid. It is believed that the overexpression of the serA gene product may increase serine concentration in the cell. Serine is one of the precursors of tryptophan. These documents also describe the use of the lacUV5 promoter to control the DS gene and the trpDCBA genes. The maximum production of tryptophan disclosed in this work is about 2400 ppm or 2.4 g/l.
The lacUV5 promoter was used by Stauffer to eliminate the natural regulation of the trp operon and aroG genes. The lacUV5 promoter is a mutated form of the lac promoter that was selected for relief of catabolite repression. The lac promoter is composed of two regions: the RNA polymerase binding site, which is composed of -10 and -35 regions, and the CAP binding site located at about the -60 region from the transcription start site. The CAP site binds the catabolite activator protein (CAP) that is responsible for activating a transcription of the lac operon when there is no glucose present. When glucose is present, CAP does not bind and transcription is not activated. There is a very low transcription from the lac promoter in the presence of glucose. This phenomenon is known as catabolite repression.
In the -10 region, the lacUV5 promoter is changed from the lac promoter by a mutation from GT to AA. This mutation in the lac promoter was found by selecting for a strain that no longer had catabolite repression of the lac operon. The reduction in catabolite repression appears to result from a better binding of the RNA polymerase to the lacUV5 promoter without the need for CAP.
The tac promoter is a combination of the lacUV5 promoter and the trp promoter using the -10 region from the lacUV5 promoter and the -35 region from the trp promoter which makes a better RNA polymerase binding site. The tac promoter no longer contains the CAP binding site, thus eliminating any catabolite repression. Under certain conditions, the tac promoter is about seven times stronger than the lacUV5 promoter. Both the lacUV5 and tac promoters still contain the lac operator region that binds the lacI gene product, the lac repressor, so both of these promoters still respond to an induction by .beta.-galactosides such as lactose and isopropyl-.beta.-D-thiogalactoside (IPTG).
Further development of the Stauffer two plasmid system and the bacterial host resulted in a one plasmid system that contains a trpEDCBA operon from E. coli including an E. coli feedback resistant AS gene, replacing the S. marcessens AS gene. The trp operon has the attenuator and promoter region removed and is controlled by the tandem lacUV5 promoter described in the Stauffer work. This plasmid, pBE7, also contains the feedback resistant aroG gene coding for the DS enzyme from one of the Stauffer plasmids under the control of the tandem lacUV5 promoters. Also contained on the pBE7 plasmid are the serA and lacI genes from the Stauffer plasmids. The invention uses various plasmids developed from the plasmid pBE7 illustrated in FIG. 1.
The host strain (JB102) was also developed from Stauffer's host strain. The host strain was developed in several steps from the bacterial strain B1238 having the genotype W3110 F' .DELTA.(lacU169, .DELTA.(gal-bio), (trp-lac)) W205 (trp-61-intc-226)!, as described by Benedik et al., Gene, 19:303-311 (1982). This strain B1238 was further developed into strain C534 by the Stauffer Chemical Company as described in WO 87/01130. Strain C534 was further developed by Genencor International by a P1 transduction using a lysate of W3110 and transducing C534 to the genotype trp+, lac-. The resulting strain, PB103, was then transduced with a P1 lysate from the strain JC158 described in Genetics (1963) 48:105-120, which is serA, and a strain was selected that was serA. This strain is JB102 and has the genotype lacU169, tna, serA, anthranilate resistant!.
The Genencor strain (JB102/pBE7) produces about 35 g/l tryptophan in fermenters.